The design, development and application of

electrochemical glutamate biosensors

G. Hughes1, R.M. Pemberton1, P.R. Fielden2, J.P. Hart1*

1. Centre for Research in Biosciences, Faculty of Health and Applied Sciences, University of

the West of England, Bristol, Coldharbour Lane, Bristol, BS16 1QY

2. Department of Chemistry, Lancaster University, Bailrigg, Lancaster, United Kingdom,

LA1 4YB

*Corresponding author: john.hart@uwe.ac.uk Tel: 0117 328 2469

Abstract

The development of biosensors for the determination of glutamate has been of great scientific

interest over the past twenty five years owing to its importance in biomedical and food studies.

This review will focus on the various strategies employed in the fabrication of glutamate

biosensors together with their performance characteristics. A brief comparison of the enzyme

immobilisation method employed, as well as the performance characteristics of a range of

glutamate biosensors are described in tabular form and then described in greater detail throughout

the review: some selected examples have been included to demonstrate the ways in which these

biosensors may be applied to real samples.

Keywords: glutamate oxidase, glutamate dehydrogenase, monosodium glutamate,

amperometry, carbon nanotubes, reagentless, biosensor, NADH, NAD+, differential pulse

voltammetry.

1. Introduction

Glutamate is considered to be the primary neurotransmitter in the mammalian brain and

facilitates normal brain function [1]. Neurotoxicity, which causes damage to brain tissue, can

be induced by glutamate at high concentrations, which may link it to a number of

neurodegenerative disorders such as Parkinson’s disease, multiple sclerosis [2] and

Alzheimer’s disease [3]. In cellular metabolism, glutamate also contributes to the urea cycle

and tricarboxylic acid cycle (TCA)/Krebs cycle. It plays a vital role in the assimilation of

NH4+ [4]. Intracellular glutamate levels outside of the brain are typically 2–5 mM/L, whilst

extracellular concentrations are ~0.05 mM/L [5]. It is also present in high concentrations

throughout the liver, kidney and skeletal muscle [6].

Glutamate is also found in many foods in the form of monosodium glutamate (MSG) as a

means to reduce salt intake and enhance flavour [7]. MSG is the source of some controversy,

despite the fact that the EU limit of MSG in foods is 10g/kg of product, it is typically found

in high concentrations in food claiming to contain no added MSG [8]. It can also be used to

mask ingredients of poor freshness. The concentration of MSG in foods can vary

significantly. The presence of monosodium glutamate in wastewater is also a concern due to

its inhibitory effects on wheat seed germination and root elongation [9].

Electrochemical biosensors for the detection of glutamate offer a faster, more user-friendly

and cheaper method of analysis in comparison to classical techniques such as high

performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry

(GC-MS). This review discusses electrochemical biosensors fabricated based on glutamate

oxidase or glutamate dehydrogenase. The method of enzyme immobilization and, where

applicable, the application of glutamate biosensors to biological and food samples.

2. Biosensors based on glutamate oxidase

In this section, the fabrication methods are sub-divided according to the technique of enzyme

immobilization. The electrochemical response can generally be described by the following

equations:

Glutamate + O2 GluOx H2O2 + α-ketoglutarate (1)

H2O2 2H+ + O2 + 2e- (2)

Equation 1 represents the enzymatic oxidation of the glutamate to form α-ketoglutarate and

H2O2. Equation 2 describes the electrochemical detection of hydrogen peroxide at the base

transducer which generates the analytical response.

2.1. Entrapment

The entrapment of enzymes is defined as the integration of an enzyme within the lattice of a

polymer matrix or a membrane, whilst retaining the protein structure of the enzyme [10]. In

addition to the immobilization of enzymes, membranes can also eliminate potential interfering

species that may be present in complex media such as serum and food.

A selective biosensor for the determination of glutamate in food seasoning was developed by

incorporating glutamate oxidase into a poly(carbamoyl) sulfonate (PCS) hydrogel. The GluOx-

PCS mixture was then drop-coated onto the surface of a thick-film platinum electrode [11].

Liquid samples (1, 10 and 100µL) were diluted to 10mL with phosphate buffer. The biosensor

was then utilised to determine the glutamate recovery from different concentrations of the

sample. The results generated correlated favourably with a L-glutamate colorimetric test kit.

A recent application of a micro glutamate biosensor for investigating artificial cerebrospinal fluid

(CSF) under hypoxic conditions was described [12]. The fabrication method is a complex, multi-

step process whereby glutamate oxidase is incorporated with chitosan and ceria-titania

nanoparticles (Figure 1).

4

Figure 1: Schematic illustration of the biosensor design and the GluA detection principle. (Reprinted with permission from [12], Elsevier)

The nanoparticles are able to store and release oxygen in its crystalline structure; it can supply O 2

to GluOx to generate H2O2 in the absence of environmental oxygen. The biosensor was evaluated

with artificial CSF which had been fortified with glutamate over the physiological range; the

device was found to operate over the concentration of interest under anaerobic conditions.

A device for the measurement of glutamate in brain extracellular fluid utilising a relatively simple

fabrication procedure has been reported [13]. The procedure involved dipping a 60-µm radius

Teflon coated platinum wire into a buffered solution containing glutamate oxidase and o-

phenylenediamine (PPD), followed by a solution containing phosphatidylethanolamine (PEA)

and bovine serum albumin (BSA). The glutamate oxidase was entrapped by the

electropolymerization of PPD on the surface of the electrode. The PPD and PEA was used to

block out interferences.

An interesting entrapment approach employing polymers to encapsulate GluOx onto a gold

electrode has been reported [14]. The first step involved the immersion of a gold disc electrode in

3-mercaptopropionic acid (MPA) solution, followed by drop-coating layers of poly-L-lysine and

poly(4-styrenesulfonate). Once dry, a mixture of GluOx and glutaraldehyde was drop-coated on

to the surface to form a bilayer. The authors suggested that MPA increases the adhesion of the

polyion complex to the gold surface by the electrostatic interaction between the carboxyl groups

5

present on the MPA and the amino groups present on the poly-L-lysine. A response time of only

3 seconds was achieved after an addition of 20nM glutamic acid, which gave a current of 0.037

nA (1.85 nA/µM). A linear response was observed between 20µM and 200µM. Both the response

time and limit of detection are superior to previously discussed biosensors. It was suggested that

the rapid response was due to the close proximity of the enzymatic reaction to the surface of the

electrode. For this method of fabrication of glutamate oxidase based biosensors, the latter

approach leads to the lowest limit of detection.

The increased interest in glutamate measurement has led to the commercial development of an in

vivo glutamate biosensor by Pinnacle Technology Inc. [15]; this has been successfully used for

monitoring of real-time changes of glutamate concentrations in rodent brain. The biosensor

employs an enzyme layer composed of GluOx and an “inner-selective” membrane, composed of

an undisclosed material that eliminates interferences. The enzymatically generated hydrogen

peroxide is monitored using a platinum-iridium electrode. The biosensor possesses a linear-range

up to 50µM. The manufacturers indicate that the miniaturised biosensor requires calibration upon

completion of an experiment in order to ensure the selectivity and integrity of the sensors.

2.2. Covalent-bonding

The application of a glutamate oxidase based biosensor for the measurement of glutamate in the

serum of healthy and epileptic patients has been described [16]. The fabrication method consists

of electrodepositing chitosan (CHIT), gold nanoparticles (AuNP) and multiwalled carbon

nanotubes (MWCNTs) on the surface of a gold electrode.

The serum sample was diluted with phosphate buffer solution (PBS) before analysis. The

concentration of the glutamate in the sample was determined using a standard calibration curve

constructed from the amperometric responses obtained with glutamate in PBS. The results

compared favourably with a colorimetric test kit. A low operating potential of +0.135 V vs.

Ag/AgCl for measuring the enzymatically generated H2O2 was significantly lower than other

6

biosensors based on GluOx [13,17], The time taken to reach 95% of the maximum steady state

response was 2 seconds after the initial injection. This method of fabrication whilst complex,

possesses a significantly lower operating potential when compared to other biosensors fabricated

using entrapment techniques.

2.3. Cross-linking

Enzyme immobilization can be achieved by intermolecular cross-linking of the protein structure

of the enzyme to other protein molecules or within an insoluble support matrix. Jamal et. al. [17]

have described a complex entrapment method which consisted of drop-coating a 10µL mixture of

GluOx (25U in 205µL), 2mg of BSA, 20µL of glutaraldehyde (2.5% w/v) and 10µL of Nafion

(0.5%) onto a platinum nanoparticle modified gold nanowire array (PtNP-NAE) and allowing it

to dry overnight under ambient conditions. The fabrication technique is illustrated in Figure 2.

The analytical response results from the oxidation of H2O2 at the gold nanowire electrode as

illustrated in Equation 2.

Figure 2: Schematic illustration of stepwise fabrication of the GlutOx/PtNP/NAEs electrodes. Reprinted

with permission from [18], Elsevier.

The high sensitivity obtained appears to be related to the presence of the nanoparticles at the gold

electrode surface. The nanoparticles act as conduction centres and facilitate the transfer of

electrons towards the gold electrode. Additionally, a high enzyme loading was utilised, which in

7

combination with the nanoparticles, resulted in increased enzyme immobilisation to the surface.

However, given the high enzyme loading and use of both a gold nanowire electrode and platinum

nanoparticles, the biosensor is unlikely to be commercially viable due to its high cost.

GluOx was immobilized to the surface of a palladium-electrodeposited screen printed carbon

strip by a simple crosslinking immobilisation technique using a photo-crosslinkable polymer

(PVA-SbQ) [18]. The biosensor exhibited a stable steady state response for six hours in a stirred

solution indicating that the enzyme was fully retained within the polymer membrane.

A glutamate biosensor [19] was successfully applied to the determination of MSG in soy sauce,

tomato sauce, chicken thai soup and chilli chicken. MSG levels compared very favourably with a

spectrophotometric method (2 - 5% CoV based on n = 5). The biosensor was fabricated by

mixing glutamate oxidase, BSA and glutaraldehyde, then spreading the mixture onto the surface

of an O2 permeable poly-carbonate membrane. The membrane was then attached to an oxygen

probe using a push cap system and oxygen consumption was measured at an applied potential of

-0.7 V; this is a considerably more negative operating potential compared to previously discussed

biosensors. In this case, the response is a result of the reduction reaction shown in Equation 3.

O2 + 2e- + 2H+ H2O2 (3)

The lowest limit of detection achieved with a glutamate biosensor was fabricated by covalently

immobilizing glutamate oxidase onto polypyrrole nanoparticles and polyaniline composite film

(PPyNPs/PANI) [20]. Cyclic voltammetry was used to co-electropolymerize the compounds onto

the surface. The PPyNPs act as an electron transfer mediator which allows a low operating

potential to be employed (+85 mVs), that reduces the likelihood of oxidising interferences. The

authors also claim that this leads to an increase in the sensitivity of the biosensor. The biosensor

was successfully applied to the determination of glutamate in food samples including tomato soup

and noodles. High recoveries of 95% - 97% were achieved which compares favourably with

values attained by previously discussed biosensors [19].

8

3. Biosensors based on glutamate dehydrogenase

This section is subdivided in a similar way to section 2, ie: according to the method of

immobilization. The electrochemical response can generally be described by the following

equations:

Glutamate + NAD+ GLDH NADH + α-ketoglutarate (4)

NADH + Mediatorox NAD+ + Mediatorred (5)

Mediatorred ne- + mH+ + Mediatorox (6)

Equation 4 represents the enzymatic reduction of the cofactor NAD+ to NADH and the oxidation

of glutamate to α-ketoglutarate. Equation 5 represents the electrochemical reduction of the oxidised

mediator to the reduced form (Mediatorred). Equation 6 describes the electrochemical oxidation of

Mediatorred at the base transducer which generates the analytical response; the regenerated

mediator ox, can then undergo further reactions with NADH. Equations 4 and 5 represent the

electrocatalytic oxidation of NADH, which occurs at lower applied potentials than obtained by

the direct electrochemical oxidation of NADH at an unmodified electrode.

3.1. Entrapment

A simple fabrication method based on the integration of a liver mitochondrial fraction containing

glutamate dehydrogenase was employed for the fabrication of a novel glutamate biosensor. The

liver mitochondrial fraction was utilised in an attempt to reduce the cost of the biosensor,

however, the activity of the biosensor appeared to be compromised in comparison to the

biosensor incorporating purified glutamate dehydrogenase. The biological recognition element

was mixed with a carbon paste, packed into an tube, and used in the determination of MSG in

chicken bouillon cubes [21]. The enzymatically generated NADH was oxidised using

ferricyanide as a electrochemical mediator. High recoveries of MSG were achieved. Several

amino acids, commonly found in food products, did not interfere with the determination.

Extensive pre-treatment of the food sample, consisting of dissolving, vacuum-filtering, washing

9

and then further diluting the sample in buffer, was required before analysis, in contrast to simpler

food preparation methods previously discussed [19].

A novel biosensor fabrication technique was developed by Tang et. al. [22] which consisted of

entrapping GLDH between layers of alternating poly(amidoamine) dendrimer-encapsulated

platinum nanoparticles (Pt-PAMAM) with multi-walled carbon nanotubes. PAMAM’s were used

to modify the surface of the glassy carbon electrode due to their excellent biocompatibility and

chemical fixation properties. The procedure was repeated using positively charged Pt-PAMAM

and negatively charged GLDH which were alternatively adsorbed onto the CNTs in a layer-by-

layer process. The assembly process and enzyme immobilisation process is illustrated in Figure 3.

Figure 3: Schematic showing the procedure of immobilizing Pt-PAMAM onto CNTs (a) and the layer-by-

layer self-assembly of GLDH and Pt-PAMAM onto CNTs (b). Pt-PAMAM/CNTs heterostructures were

attached covalently via EDC, Pt-PAMAM and GLDH were alternately deposited. Reprinted with permission

from [24], Elsevier.

Whilst the fabrication procedure is complex, the biosensor possesses superior sensitivity to

previously discussed biosensors.

10

In contrast to the above, a simpler and less time consuming method of fabricating a glutamate

biosensor has been described [23] which involved incorporating GLDH and NAD+ into carbon

paste. The mixture was inserted in a holder, placed in to a solution containing O-

phenylenediamine and subsequently electropolymerized. The o-phenylenediamine film is

simultaneously able to prevent interferences and facilitate the amperometric detection of NADH

at low applied potentials by acting as an electron mediator. The biosensor was used to determine

glutamate in chicken bouillon cubes; the results compared favourably to those obtained using a

spectrophotometric method (12.6 ± 0.3% (n = 5) and 12.3% respectively). Despite the simpler

fabrication method, the linear range and sensitivity of the biosensor was lower than the sensor

described by Tang et. al. [22].

In recent years, the biopolymer known as chitosan has been investigated in a variety of studies as

a method of entrapping enzymes for biosensor construction [24]. CHIT appears to offer improved

enzyme stability and is easy to immobilize onto a variety of materials. A biosensor for glutamate

[25] has been constructed by depositing a mixture of purified CNTs, CHIT and MB onto a glassy

carbon electrode and dried. The surface was then treated with an aliquot of GLDH in PBS, and

dried at 4ºC. The cofactor was present in free solution at a concentration of 4mM. The selectivity

of the biosensor was determined by the addition of interferences (AA, UA), with no apparent

amperometric responses being generated. However, the application of the biosensor to clinical

and food samples was not described in this paper.

Screen-printing technology has offered the possibility of the mass production at low cost of

glutamate biosensors; these have been successfully applied to the measurement of glutamate in

serum and food samples [26]. As screen-printed devices are inexpensive to manufacture they can

be considered disposable, in comparison to glassy carbon electrodes, which are expensive and are

not considered disposable devices. Consequently, the former are much convenient devices for

fabricating biosensors and have become a popular route to commercialisation. The fabrication

method involved drop-coating CHIT (0.05%) onto the surface of a Meldola’s Blue (MB) SPCE

11

(MB-SPCE), followed by an aliquot of glutamate dehydrogenase (3U/µL). This device is

designated GLDH-CHIT-MB-SPCE. The biosensor was then left to dry under vacuum at 4ºC

overnight. NAD+ was present in free solution at a concentration of 4mM.

The electro-catalyst Meldola’s Blue allowed an operating potential of only +100mV to be

employed; the response occurred as a result of the electrocatalytic oxidation of enzymatically

generated NADH. The biosensor was successfully applied to the determination of MSG in Beef

OXO cubes and endogenous glutamate in serum. The beef OXO cube was dissolved by

sonication in PBS and the endogenous content of both the OXO cube and serum were

determined. Recoveries of 91% were attained for the spiked OXO cube (n = 6) and 96% for the

spiked serum test (n = 6). These results compare favourably to those reported by Alvarez-Crespo

et. al. [23] and Basu et.al [19]. This device improves upon the linear range of previously

discussed biosensors, [23,25].

In order to further develop this biosensor for potential commercial development, all of the

components needed to be immobilized onto the surface of the transducer. The layer-by-layer

fabrication process is described in the paper [27]. The schematic shown in Figure 4 summarises

the important steps involved in this process.

12

Figure 4: A schematic diagram displaying the layer-by-layer drop coating fabrication procedure used to

construct the reagentless glutamate biosensor, based on a MB-SPCE electrode. Reprinted with

permission from [27], Elsevier.

An amperometric plot using the reagentless biosensor are shown in Figure 5. The biosensor was

successfully applied to the determination of glutamate in spiked serum. A recovery of 104% (n =

5, CoV: 2.91%) was determined, which compares favourably to previously discussed biosensors

[19,23,26]. An interference study was conducted in both serum and food samples (stock cubes)

and no interfering signals were generated. Such reagentless biosensors offer the advantage of

being low cost, simple to use and requires no additional cofactor to be added to the sample

solution. Clearly, this is a prerequisite for commercial devices.

13

Figure 5: Amperogram conducted with the reagentless glutamate biosensor. Each arrow represents an

injection of 3 μL of 25 mM glutamate in a 10 mL stirred solution containing supporting electrolyte; 75 mM,

PB (pH 7.0), with 50 mM NaCl at an applied potential of +0.1 V vs. Ag/AgCl. Adapted with permission from

[27], Elsevier.

3.2. Covalent Bonding

The electrochemical technique known as different pulse voltammetry was used in conjunction

with a glutamate biosensor to develop a novel assay for measuring glutamate in a mixture of

naturally occurring biomolecules commonly found in biological samples [28]. The biosensor

fabrication procedure involves vertically aligned carbon nanotubes (VACNTs) which are treated

in order to convert the tips of the CNTs into carboxylic acid groups, in order to covalently bind

the enzyme. GLDH was bound to the CNTs using 1-(3-dimethylamino propyl)-3-

14

ethylcarbondiimide hydrochloride (EDC) and hydroxyl-sulfosuccinimide sodium salt (sulfo-

NHS) to promote amide linkages between the carboxylic tips of the CNTs and the lysine residue

present on the enzyme. This was achieved by immersing the electrode in a solution containing

EDC/sulfo-NHS and mixed. Once dried, the enzyme was drop-coated onto the electrode, dried

for 2 hours and washed with PBS/BSA mixture. The DPV obtained with the synthetic samples

indicated that over the concentration range studied, no significant interferences should be

expected. However, the biosensor was not applied to a real sample.

3.3. Crosslinking

Carbon nanotubes have been successfully employed to produce cross linking matrixes. Single

wall carbon nanotubes have been treated with thionine (Th) to produce a Th-SWCNT

nanocomposite on the surface of a glassy carbon electrode [29]. The nanocomposite acts as both

an electron mediator and enzyme immobilization matrix. The GLDH was mixed with BSA and

crossed linked with glutaraldehyde and coated onto the Th-SWCNT layer. An applied potential of

+190mV was utilised for amperometric measurements. The linear range was found to superior to

that which was achieved by Gholiazadeh et. al. [28] and Tang et. al. [22]. Ascorbic acid, uric acid

and 4-acetamidophenol were examined as possible interferents; no discernible current responses

were seen.

15

4. Conclusions

The review has highlighted some novel approaches to the fabrication of electrochemical

glutamate biosensors. The performance characteristics of the glutamate biosensors discussed are

summarised within Table 1. The most commonly reported method for the immobilization of both

glutamate oxidase and dehydrogenase is entrapment.

The utilisation of glutamate oxidase offers an advantage over glutamate dehydrogenase as the

latter requires the cofactor NAD+ to be co-immobilised onto the appropriate transducer. In

addition the response times are also generally shorter for glutamate oxidase based biosensors.

However, there are several drawbacks with the use of these biosensors. Significantly higher

applied potentials must be utilised in order to generate an amperometric response from the

enzymatic generation of H2O2. In contrast low operational potentials of around 0 V can be applied

with dehydrogenase based biosensors; which is advantageous when measuring glutamate in

complex media.

Expensive electrode materials such as gold and platinum are often used in the development of

glutamate biosensors utilising glutamate oxidase. In contrast, three glutamate biosensors

employing GLDH immobilized onto SPCE’s as the electrode material have been described.

SPCE’s offer an inexpensive approach to fabricating glutamate biosensors which is clearly an

important consideration in the commercialisation of such devices.

Finally, the cost of glutamate dehydrogenase is far lower than that of glutamate oxidase (as of

August 2015). 100mg (20U per mg) of glutamate dehydrogenase costs £175. By contrast,

glutamate oxidase is sold by Sigma for £88.20 per 1U. This is clearly an important consideration

for commercialisation.

16

Table 1: Details of the enzyme immobilisation technique and performance characteristics of amperometric

glutamate biosensors based on glutamate oxidase and glutamate dehydrogenase

Legend: NS – Not specified, N/A – Not applicable * - Voltametric Measurement, ~ - Polarographic Measurement

Glutamate Oxidase based single enzyme systemsImmobilisation Technique Type Ref LOD Optimum

pHV (vs. Ag/AgCl) Sensitivity Linear

Range Stability Response Time

Poly(carbamoylsulphonate) (PCS) hydrogel mixed with GluOx

Entrapment [11] 1.01µM 6.86 +400mV 1.94 nA/µM 100 - 5000µM

90% of activity retained after 2 weeks.

NS

Mixed ceria and titania nanoparticles for the detection of glutamate in hypoxic environments

Entrapment [12] 0.594µM 7.4 +600mV

0.7937 nA/µM 5 – 50µM

75% of signal after 70 assays in aerobic environment.Anaerobic enviroment, stable for 8 assays.

~5

Biosensor for Neurotransmitter L-Glutamic Acid Designed for Efficient Use of L-Glutamate Oxidase and Effective Rejection of Interferences

Entrapment [13] 0.27 µM 7.4 +700 mV 3.8 ± 1.3 nA cm-2 mmol-1

L

0 - 100µM NS <10s

17

Polyion complex-bilayer membrane Entrapment [14] 0.2 nM 7.0 +800 mV 1.85 nA/µM 3 – 500 µM

Stable over 4 weeks with daily usage.

NS

Carboxylated multiwalled carbon nanotubes/gold nanoparticles/chitosan composite film modified Au electrode

Covalent-bonding [16] 1.6µM 7.4 +135mV 155

nA/µM/cm2 5 – 500 μM 4 months, no data shown. 2s

Pt nanoparticles modified Au nanowire array electrode Entrapment [17] 14µM 7.4 +650mV 194.6 µA

mM-1 cm-2200 - 800 µM

98% of its initial response retained after 2 weeks.

4.8s

Photo-crosslinkable polymer membrane on a palladium-deposited screen-printed carbon electrode

Cross linking [18] 0.05µM 7.0 +400mV 12.8 nA/µM 0.05µM - 100µM

Stored dry – 5 months, no change in response.

30 – 50sNot stated.

Cross linking with Glutaraldehyde and BSA as a spacing agent. ~

Cross linking [19] N/A 7.0 -700mV NS NS

84% of initial signal after 48 days of use every 3 days.

120s

Polypyrrole nanoparticles/polyaniline modified gold electrode.

Cross linking [20] 0.1nM 7.5 -130mV 533 nA/ µM/cm2

0.02 - 400µm 60 days at 4ºC 3s

18

Glutamate Dehydrogenase based single enzyme systems

Immobilisation Technique Type Ref LOD pHV (vs. Ag/AgCl)

[NAD+/NAD(P)+]

Sensitivity Linear Range Stability Response

Time

A mixture of carbon paste, octadecylamine and mitochondria fraction, packed into the working electrode.

Entrapment [21] 100µM 7.5 +350mV 1mM 0.189µA/mM 0.4 – 10mM

60% after 10 days of use.

N/A

Alternatively assembling layers of glutamate dehydrogenase and Pt-PAMAM.

Entrapment [22] 100 µM 7.4 +200mV 0.1mM 433 µA/mM-1

cm20.2 - 250 µM

85% after four weeks

3s

Unmodified carbon paste mixed with lyophilized enzyme and coenzyme, packed into the well of a working electrode.

Entrapment [23] 3.8µM 8.0 +150 mV N/A 4·6×105 nA l mol−1

5 - 78 µM

Signal decrease by 50% after 3 days

120s

GLDH dropcoated onto the surface of a CNT-CHIT-MDB modified GC electrode.

Entrapment [25] 2 µM 7.0 -140mV 4mM 0.71 ± 0.08 nA/μM

25 – 100 µM NS NS

19

Immobilisation of GLDH with CHIT, drop coated onto the surface of a SPCE.

Entrapment [26] 1.5 µM 7.0 +100mV 4mM 0.44nA/µM 12.5 - 150µM NS 2s

Reagentless biosensor. Immobilisation of both NAD+ and GLDH by utilising a mixture CHIT/MWCNT/MB dropcoated onto the surface of a SPCE in a layer by layer fashion.

Entrapment [27] 3µM 7.0 +100mV N/A 0.39 nA/µM 7.5 - 105µM

100% of original response retained after 2 weeks

20 – 30s

GLDH attached to CNTs utilising EDC and sulfo-NHS, by immersing electrode in PBS containing both compounds then dropcoating GLDH. *

Covalent bonding [28] 0.57

µM 7.4 NS 2mM

With mediator:1.17 mA mM−1 cm−2 / 0.153 mA mM−1 cm−2

Without mediator:0.976 mA mM−1 cm−2 / 0.182 mA mM−1 cm−2

With mediator:0.1 - 20µM / 20 - 500µM

Without mediator0.1 - 20µM / 20 - 300µM

80.5% of original response after two weeks of use.

NS

GLDH is mixed with BSA then coated onto the surface of a Th-SWNTs/GC electrode, then followed by glutaraldehyde.

Crosslinking [29] 0.1 µM 8.3 +190mV vs NHE 0.1µM 137.3 µA

mM-10.5 – 400µM

93% after 2 weeks 5s

20

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